Aqueous ophthalmic solution

ABSTRACT

Provided is an aqueous ophthalmic solution comprising diquafosol or a salt thereof at a concentration of 0.1% to 10% (w/v) and a chlorhexidine at a concentration of 0.0001% to 0.1% (w/v).

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/539,283, filed on Jun. 23, 2017, now U.S. Pat. No. 10,278,985, whichis a U.S. National Stage of International Application No.PCT/JP2015/086230, filed on Dec. 25, 2015, which in turn claims priorityto Japanese Application No. 2015-114595, filed on Jun. 5, 2015, andJapanese Patent Application No. 2014-263003, filed on Dec. 25, 2014, theentire contents of each of which is incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to an aqueous ophthalmic solutioncomprising diquafosol or a salt thereof at a concentration of 0.1% to10% (w/v) (to also be referred to as diquafosol ophthalmic solution),which also comprises a chlorhexidine at a concentration of 0.0001% to0.1% (w/v).

BACKGROUND ART

Ophthalmic solutions typically consist of types that are used multipletimes over a fixed period of time after opening (multi-dose typeophthalmic solutions) and types that are used only once (unit-dose typeophthalmic solutions). In particular, multi-dose type ophthalmicsolutions typically comprise preservatives to prevent decomposition ofthe product due to microbial contamination during use and ensurepreservation stability.

Benzalkonium chloride (to also be referred to as BAK) is commonly usedas a preservative that has superior preservative effects. On the otherhand, the use of BAK at a high concentration is known to have thepotential to cause corneal disorders. In addition, if a user applies anophthalmic solution comprising BAK while wearing soft contact lenses,BAK has been indicated to have a detrimental effect on the soft contactlenses such as by causing deformation thereof as a result of makingcontact therewith. Consequently, the application of an ophthalmicsolution comprising BAK is normally prohibited when wearing soft contactlenses.

Diquafosol is a purine receptor agonist, also referred to asP¹,P⁴-di(uridine-5′)tetraphosphate or Up₄U, that is used for thetreatment of dry eye in Japan in the form of an ophthalmic solutioncomprising diquafosol sodium at a concentration of 3% (w/v) (trade name:Diquas® Ophthalmic Solution 3%). This Diquas® Ophthalmic Solution 3%comprises BAK for the reason indicated above.

In addition, Patent Document 1 discloses an aqueous ophthalmic solutioncomprising diquafosol or a salt thereof at a concentration of 0.1% to10% (w/v) and a chelating agent at a concentration of 0.0001% to 1%(w/v).

PRIOR ART DOCUMENTS Patent Documents

-   [Patent Document 1] Japanese Unexamined Patent Application    Publication No. 2013-227291

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

An object of the present invention is to provide a diquafosol ophthalmicsolution that demonstrates a higher degree of safety without comprisingBAK.

Means for Solving the Problems

As a result of conducting extensive studies to solve the aforementionedproblems, the inventors of the present invention found that an aqueousophthalmic solution comprising diquafosol or a salt thereof at aconcentration of 0.1% to 10% (w/v) and a chlorhexidine at aconcentration of 0.0001% to 0.1% (w/v) (to be referred to as the presentophthalmic solution) has superior preservative effects, thereby leadingto completion of the present invention. Moreover, the inventors of thepresent invention found that the present ophthalmic solution preventsdeformation of a soft contact lens. In addition, the inventors of thepresent invention found that the present ophthalmic solutiondemonstrates a higher level of cell viability in cultured immortalizedhuman corneal epithelial cells than ophthalmic solutions comprising BAKand ophthalmic solutions comprising chlorhexidine but not comprisingdiquafosol or a salt thereof. In addition, the inventors of the presentinvention found that the present ophthalmic solution significantlyincreases non-invasive tear break up time (NIBUT) in an eye wearing asoft contact lens, or in other words, stabilizes the tear film in an eyewearing a soft contact lens. On the other hand, such effects have notbeen observed with artificial tear solutions. Since the onset and/orexacerbation of dry eye symptoms caused by wearing soft contact lensesare attributable to a decrease in tear film stability, stabilization ofthe tear film by the present ophthalmic solution is useful for theprevention and/or treatment of dry eye in an eye wearing a soft contactlens. In addition, the present ophthalmic solution is also useful forthe prevention and/or treatment of eye dryness and/or eye discomfort inan eye wearing a soft contact lens.

Namely, the present invention provides an aqueous ophthalmic solution asindicated below.

(1) An aqueous ophthalmic solution comprising diquafosol or a saltthereof at a concentration of 0.1% to 10% (w/v) and a chlorhexidine at aconcentration of 0.0001% to 0.1% (w/v).

(2) The aqueous ophthalmic solution described in (1), wherein thechlorhexidine is chlorhexidine gluconate.

(3) The aqueous ophthalmic solution described in (1) or (2), wherein theconcentration of the chlorhexidine in the ophthalmic solution is 0.0005%to 0.05% (w/v).

(4) The aqueous ophthalmic solution described in (1) or (2), wherein theconcentration of the chlorhexidine in the ophthalmic solution is 0.001%to 0.005% (w/v).

(5) The aqueous ophthalmic solution described in any of (1) to (4),wherein the concentration of diquafosol or a salt thereof in theophthalmic solution is 3% (w/v).

(6) The aqueous ophthalmic solution described in any of (1) to (5),further comprising a chelating agent.

(7) The aqueous ophthalmic solution described in (6), wherein thechelating agent is edetic acid or a salt thereof.

(8) The aqueous ophthalmic solution described in any of (1) to (7),which is for a soft contact lens.

(9) The aqueous ophthalmic solution described in (8), wherein the softcontact lens is a silicone hydrogel contact lens.

In addition, the present invention provides a method for preventingdeformation of a soft contact lens as indicated below.

(10) A method for preventing deformation of a soft contact lens with theaqueous ophthalmic solution described in any of (1) to (7).

Moreover, the present invention also relates to that indicated below.

(11) An aqueous ophthalmic solution for the prevention and/or treatmentof dry eye comprising diquafosol or a salt thereof at a concentration of0.1% to 10% (w/v) and a chlorhexidine at a concentration of 0.0001% to0.1% (w/v).

(12) A use of an aqueous ophthalmic solution comprising diquafosol or asalt thereof at a concentration of 0.1% to 10% (w/v) and a chlorhexidineat a concentration of 0.0001% to 0.1% (w/v) for the prevention and/ortreatment of dry eye.

(13) A method for the prevention and/or treatment of dry eye, comprisingthe application of an aqueous ophthalmic solution comprising diquafosolor a salt thereof at a concentration of 0.1% to 10% (w/v) and achlorhexidine at a concentration of 0.0001% to 0.1% (w/v).

In addition, the present invention provides the aqueous ophthalmicsolutions indicated below.

(14) The aqueous ophthalmic solution described in any of (1) to (7) forthe prevention and/or treatment of dry eye in an eye wearing a softcontact lens. (15) The aqueous ophthalmic solution described in any of(1) to (7) for improving tear film stability in an eye wearing a softcontact lens.

(16) The aqueous ophthalmic solution described in any of (1) to (7) forthe prevention or treatment of eye dryness or eye discomfort in an eyewearing a soft contact lens.

(17) The aqueous ophthalmic solution described in any of (14) to (16),wherein the soft contact lens is a silicone hydrogel contact lens.

Effects of the Invention

As is clear from the results of test examples to be subsequentlydescribed, the present ophthalmic solution has superior preservativeeffects. Moreover, since the present ophthalmic solution preventsdeformation of a soft contact lens, it can be used for a soft contactlens. In addition, the present ophthalmic solution demonstrates a higherlevel of cell viability in cultured immortalized human cornealepithelial cells than ophthalmic solutions comprising BAK or ophthalmicsolutions comprising chlorhexidine but not comprising diquafosol or asalt thereof. Accordingly, the present ophthalmic solution demonstrateshigher safety with respect to a living body, and particularlykeratoconjunctival epithelium, and is useful for use in diseases such asdry eye in which the keratoconjunctival epithelium is unstable. Inaddition, the present ophthalmic solution significantly increases NIBUTin an eye wearing a soft contact lens. On the other hand, such effectsare not observed with artificial tear solutions. Namely, the presentophthalmic solution stabilizes the tear film in an eye wearing a softcontact lens. Since the onset and/or exacerbation of dry eye symptomscaused by wearing soft contact lenses is attributable to a decrease intear film stability, stabilization of the tear film by the presentophthalmic solution is useful for the prevention and/or treatment of dryeye in an eye wearing a soft contact lens. In addition, the presentophthalmic solution is also useful for the prevention and/or treatmentof eye dryness and/or eye discomfort in an eye wearing a soft contactlens.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the results of a cytotoxicity test usingcorneal epithelial cells in Test Example 3.

FIG. 2 is a graph showing the results of an Evaluation Test 1 of NIBUTincreasing effect in Test Example 4.

FIG. 3 is a graph showing the results of an Evaluation Test 2 of NIBUTincreasing effect in Test Example 5.

DESCRIPTION OF THE EMBODIMENTS

The following provides a more detailed explanation of the presentinvention.

Diquafosol is a compound represented by the chemical structural formulaindicated below.

There are no particular limitations on the diquafosol salt provided itis a salt that is acceptable for use as a pharmaceutical, and examplesthereof include metal salts such as lithium, sodium, potassium, calcium,magnesium or zinc salt, salts of an inorganic acid such as hydrochloricacid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid orphosphoric acid, salts of an organic acid such as acetic acid, fumaricacid, maleic acid, succinic acid, citric acid, tartaric acid, adipicacid, gluconic acid, glucoheptonic acid, glucuronic acid, terephthalicacid, methanesulfonic acid, lactic acid, hippuric acid,1,2-ethanedisulfonic acid, isethionic acid, lactobionic acid, oleicacid, pamoic acid, polygalacturonic acid, stearic acid, tannic acid,trifluoromethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonicacid, lauryl sulfate ester, methyl sulfate, naphthalenesulfonic acid orsulfosalicylic acid, quaternary ammonium salts such as methyl bromide ormethyl iodide, salts of a halogen ion such as bromine ion, chlorine ionor iodine ion, salts of ammonia, and salts of an organic amine such astriethylenediamine, 2-aminoethanol, 2,2-iminobis(ethanol),1-deoxy-1-(methylamino)-2-D-sorbitol,2-amino-2-(hydroxymethyl)-1,3-propanediol, procaine orN,N-bis(phenylmethyl)-1,2-ethanediamine.

In the present invention, hydrates and organic solvates of diquafosol(free form) or a salt thereof are included in the diquafosol or a saltthereof.

In the case diquafosol or a salt thereof has crystal polymorphism andcrystal polymorphic groups (crystal polymorphic systems), these crystalpolymorphs and crystal polymorphic groups (crystal polymorphic systems)are also included in the scope of the present invention. Here, a crystalpolymorphic group (crystal polymorphic system) refers to individualcrystalline forms at each stage in the case of undergoing a change incrystalline form according to conditions and state, such as with respectto crystal production, crystallization, or storage and the like, as wellas the entire process thereof.

The diquafosol or a salt thereof of the present invention is preferablya sodium salt of diquafosol, and is particularly preferably a diquafosoltetrasodium salt (herein after, also be simply referred to as“diquafosol sodium”) represented by the chemical structural formulaindicated below.

Diquafosol or a salt thereof can be produced according to, for example,the method disclosed in Japanese Translation of PCT InternationalApplication Publication No. 2001-510484.

Although the present ophthalmic solution can also comprise an activeingredient other than diquafosol or a salt thereof, it preferablycomprises diquafosol or a salt thereof as the only active ingredientthereof.

Although the concentration of diquafosol or a salt thereof in thepresent ophthalmic solution is 0.1% to 10% (w/v), it is preferably 1% to10% (w/v) and particularly preferably 3% (w/v).

In the present invention, an aqueous ophthalmic solution refers to anophthalmic solution that uses water as a solvent (base).

In the present invention, a chlorhexidine includes chlorhexidine andsalts thereof. Chlorhexidine is a compound represented by the followingchemical structural formula that is also referred to as1,1′-hexamethylenebis [5-(4-chlorophenyl)biguanide].

In the present invention, among the aforementioned chlorhexidines, thereare no particular limitations on the chlorhexidine salt provided it is asalt that is acceptable for use as a pharmaceutical, and specificexamples thereof include organic acid salts [such as monocarboxylates(such as acetates, trifluoroacetates, butyrates, palmitates orstearates), polycarboxylates (such as fumarates, maleates, succinates ormalonates), oxycarboxylates (such as gluconates, lactates, tartrates orcitrates), and organic sulfonates (such as methanesulfonates,toluenesulfonates or tosylates)], inorganic acid salts (such ashydrochlorides, sulfates, nitrates, hydrobromides or phosphates), saltsof organic bases (such as salts of organic amines such as methyl amine,triethylamine, triethanolamine, morpholine, piperazine, pyrrolidone,tripyridine or picoline), and salts of inorganic bases [such as ammoniumsalts, alkaline metals (such as sodium or potassium), alkaline earthmetals (such as calcium or magnesium) or metals such as aluminum]. Amongthese salts, organic acid salts and/or inorganic acid salts arepreferable, oxycarboxylates, monocarboxylates and/or inorganic acidsalts are more preferable, gluconates, acetates and/or hydrochloridesare even more preferable, and gluconates are particularly preferable.One type of these chlorhexidine salts may be used alone or two or moretypes may be arbitrarily combined and used.

Chlorhexidine and salts thereof may be synthesized according to a knownmethod and can be acquired in the form of commercially availableproducts.

Although the concentration of chlorhexidine in the present ophthalmicsolution is 0.0001% to 0.1% (w/v), it is preferably 0.0005% to 0.05%(w/v) and particularly preferably 0.001% to 0.005% (w/v).

In the present invention, there are no particular limitations on the“chelating agent” provided it is a compound that chelates metal ions,and examples thereof include edetic acid or a salt thereof such asedetic acid (ethylenediamine tetraacetic acid), monosodium edetate,disodium edetate, trisodium edetate or tetrasodium edetate, dipotassiumedetate, tripotassium edetate, tetrapotassium edetate, citric acid or asalt thereof such as citric acid, monosodium citrate, disodium citrate,trisodium citrate, monopotassium citrate, dipotassium citrate ortripotassium citrate, metaphosphoric acid or a salt thereof such asmetaphosphoric acid, sodium metaphosphate or potassium metaphosphate,pyrophosphoric acid or a salt thereof such as pyrophosphoric acid,tetrasodium pyrophosphate or tetrapotassium pyrophosphate,polyphosphoric acid or a salt thereof such as polyphosphoric acid,sodium polyphosphate or potassium polyphosphate, malic acid or a saltthereof such as monosodium malate, disodium malate, monopotassium malateor dipotassium malate, tartaric acid or a salt thereof such as sodiumtartrate, potassium tartrate or potassium sodium tartrate, and phyticacid or a salt thereof such as sodium phytate or potassium phytate.Furthermore, in the present invention, edetic acid, citric acid,metaphosphoric acid, pyrophosphoric acid, polyphosphoric acid, malicacid, tartaric acid, phytic acid and salts thereof include hydrates andorganic solvates of their respective free forms or salts thereof.

In the present invention, the chelating agent is preferably edetic acid,a salt of edetic acid (edetate), citric acid, a salt of citric acid(citrate), metaphosphoric acid, a salt of metaphosphoric acid(metaphosphate), polyphosphoric acid or a salt of polyphosphoric acid(polyphosphate), and is particularly preferably a sodium salt of edeticacid (including hydrates such as disodium edetate hydrate), citric acid(including hydrates such as citric acid monohydrate), a sodium salt ofmetaphosphoric acid (sodium metaphosphate) or a sodium salt ofpolyphosphoric acid (sodium polyphosphate).

In the present invention, the salt of edetic acid is most preferablydisodium edetate hydrate (to also be simply referred to as “sodiumedetate hydrate”).

In addition, one type of these chelating agents may be used alone or twoor more types may be arbitrarily combined and used.

Although the concentration of chelating agent in the present ophthalmicsolution is, for example, 0.0001% to 1% (w/v), it is preferably 0.0005%to 0.5% (w/v) and particularly preferably 0.001% to 0.1% (w/v).

A nonionic surfactant can be incorporated in the aqueous ophthalmicsolution of the present invention as necessary. There are no particularlimitations on the nonionic surfactant provided it is within the scopeof that which is allowed as a pharmaceutical, and examples thereofinclude polyoxyethylene fatty acid esters, polyoxyethylene sorbitanfatty acid esters, polyoxyethylene castor oil derivatives,polyoxyethylene polyoxypropylene glycols and sucrose fatty acid esters.Examples of polyoxyethylene fatty acid esters include polyoxyl 40stearate, examples of polyoxyethylene sorbitan fatty acid esters includePolysorbate 80, Polysorbate 60, Polysorbate 40, polyoxyethylene sorbitanmonolaurate, polyoxyethylene sorbitan trioleate and Polysorbate 65,examples of polyoxyethylene castor oil derivatives includepolyoxyethylene (10) hydrogenated castor oil, polyoxyethylene (40)hydrogenated castor oil, polyoxyethylene (50) hydrogenated castor oil,polyoxyethylene (60) hydrogenated castor oil, polyoxyl 5 castor oil,polyoxyl 9 castor oil, polyoxyl 15 castor oil, polyoxyl 35 castor oiland polyoxyl 40 castor oil, and examples of polyoxyethylenepolyoxypropylene glycols include polyoxyethylene (160) polyoxypropylene(30) glycol, polyoxyethylene (42) polyoxypropylene (67) glycol,polyoxyethylene (54) polyoxypropylene (39) glycol, polyoxyethylene (196)polyoxypropylene (67) glycol and polyoxyethylene (20) polyoxypropylene(20) glycol.

In the present invention, an example of the nonionic surfactant is apolyoxyethylene sorbitan fatty acid ester, and is preferably Polysorbate80, Polysorbate 60, Polysorbate 40, polyoxyethylene sorbitanmonolaurate, polyoxyethylene sorbitan trioleate or Polysorbate 65, andparticularly preferably Polysorbate 80.

In addition, one type of these nonionic surfactants may be used alone ortwo or more types may be arbitrarily combined and used.

Although the concentration of nonionic surfactant in the presentophthalmic solution is, for example, 0.0001% to 10% (w/v), it ispreferably 0.0005% to 1% (w/v) and particularly preferably 0.0005% to0.1% (w/v).

A pharmaceutically acceptable additive can be added to the presentophthalmic solution as necessary using commonly used technology, and forexample, buffering agents such as sodium phosphate, sodium hydrogenphosphate, sodium dihydrogen phosphate, sodium acetate or ε-aminocaproicacid, or isotonic agents such as sodium chloride, potassium chloride orconcentrated glycerin, can be selected and added as necessary.

Although the pH of the present ophthalmic solution is only required tobe within a range allowed for ophthalmic preparations, normally it ispreferably within the range of 4 to 8. A pH adjuster such ashydrochloric acid or sodium hydroxide can be suitably added to thepresent ophthalmic solution.

The present ophthalmic solution can also be used when wearing a softcontact lens as an ophthalmic solution for a soft contact lens. Examplesof soft contact lenses include contact lenses having hydroxyethylmethacrylate for the main component thereof and silicone hydrogelcontact lenses.

There are no particular limitations on the type of soft contact lenstargeted for application of the present ophthalmic solution, and may beionic or nonionic or hydrous or non-hydrous soft contact lenses. Forexample, the present ophthalmic solution can be applied to repeatedlyusable contact lenses, one-day disposable contact lenses, one-weekdisposable contact lenses, two-week disposable contact lenses and allother soft contact lenses currently available on the market or softcontact lenses to be available on the market in the future.

Although usage of the present ophthalmic solution can be suitablyaltered corresponding to the dosage form, severity of symptoms, age andbody weight of the patient to which it is to be administered, and thediscretion of the physician, in the case of, for example, havingselected eye drops for the dosage form, the eye drops are administeredlocally to the eyes 1 to 10 times per day, preferably 2 to 8 times perday and more preferably 4 to 6 times per day.

Dry eye is defined as a chronic disease of the tears andkeratoconjunctival epithelium caused by various factors that isassociated with eye discomfort and vision disorders, andkeratoconjunctivitis sicca (KCS) is included in dry eye. In the presentinvention, the onset of symptoms of dry eye caused by wearing softcontact lenses is included in dry eye.

Symptoms of dry eye include subjective symptoms such as eye dryness, eyediscomfort, eye fatigue, heavy eyes, photophobia, eye pain or blurredvision (bleariness), as well as objective symptoms such as hyperemia orkeratoconjunctival epithelial disorders.

Although there are many aspects of the cause of dry eye that areunclear, reported examples of causes thereof include Sjogren's syndrome;congenital alacrima; sarcoidosis; graft versus host disease (GVHD)associated with bone marrow transplantation; ocular pemphigoid;Stevens-Johnson syndrome; lacrimal duct obstruction caused by trachomaor the like; diabetes; the decrease in reflex tear secretion caused bycorneal refractive surgery (laser-assisted in situ keratomileusis(LASIK)) or the like; Meibomian gland dysfunction; oily layer reductioncaused by blepharitis or the like; incomplete winking or incompleteeyelid closure caused byexophthalmos, lagophthalmos or the like; thedecrease in the secretion of mucin from a goblet cell; visual displayterminal (VDT) work, and so on.

The present ophthalmic solution can be used for the prevention and/ortreatment of dry eye.

In the present invention, the prevention and/or treatment of dry eye isdefined as the prevention and/or treatment or amelioration of thepathological symptoms and/or findings associated with dry eye, andrefers to not only the prevention and/or treatment or amelioration ofsubjective symptoms such as eye dryness, eye discomfort, eye fatigue,heavy eyes, photophobia, eye pain or blurred vision (bleariness), whichare associated with dry eye, but also includes the prevention and/ortreatment or amelioration of hyperemia associated with dry eye,keratoconjunctival epithelial disorders or the like. In addition, theprevention and/or treatment of dry eye includes the prevention and/ortreatment or amelioration of dry eye symptoms by improving tear filmstability in an eye wearing a soft contact lens. Furthermore, theprevention and/or treatment or amelioration of dry eye symptoms refersto the prevention and/or treatment or amelioration of dry eye symptomsderived from exacerbation resulting from the wearing of soft contactlenses by a patient with dry eye, and the prevention and/or treatment oramelioration of dry eye symptoms derived from the wearing of softcontact lenses per se.

In the present invention, improvement of tear film stability refers toqualitative or quantitative improvement of lacrimal fluid. Furthermore,tear film stability can be confirmed by measuring tear break up time(BUT). BUT measured in a more natural state without applying a load inthe form of a dye solution and the like is referred to as non-invasivetear break up time (NIBUT).

In the present invention, the prevention and/or treatment of eye drynessor eye discomfort in an eye wearing a soft contact lens refers to theprevention or treatment of eye dryness or eye discomfort associated withdestabilization of the tear film caused by wearing soft contact lensesor the prevention or treatment of eye dryness or eye discomfort causedby keratoconjunctival epithelial disorders attributable to theaforementioned destabilization. [Examples]

Although the following indicates the results of a preservativeeffectiveness test, deformation prevention evaluation test of softcontact lenses, cytotoxicity test using corneal epithelial cells andevaluation test of NIBUT increasing effect, these examples are providedfor the purpose of enhancing understanding of the present invention, andare not intended to limit the scope of the present invention.Furthermore, CL is the abbreviation for contact lenses while SCL is theabbreviation for soft contact lenses.

TEST EXAMPLE 1 Preservative Effectiveness Test

A preservative effectiveness test was carried out on OphthalmicSolutions 1 to 6 having the formulations shown in Table 1.

(Sample Preparation)

Ophthalmic Solution 1:

Ophthalmic Solution 1 was prepared in accordance with the formulationshown in Table 1. More specifically, diquafosol sodium (3 g), sodiumhydrogen phosphate hydrate (0.2 g), sodium chloride (0.39 g), potassiumchloride (0.15 g), sodium edetate hydrate (0.01 g), Polysorbate 80(0.0005 g) and chlorhexidine gluconate (0.002 g) were dissolved in waterand brought to a final volume of 100 mL followed by the addition of a pHadjuster to adjust to pH 7.2.

Ophthalmic Solutions 2-6:

Each of the ophthalmic solutions of Ophthalmic Solutions 2 to 6 wasprepared in the same manner as Ophthalmic Solution 1 in accordance withthe formulations shown in Table 1.

TABLE 1 (Units: g/100 ml) Ophthalmic Ophthalmic Ophthalmic OphthalmicOphthalmic Ophthalmic Solution 1 Solution 2 Solution 3 Solution 4Solution 5 Solution 6 Diquafosol sodium 3 3 3 3 3 3 Sodium hydrogen 0.20.2 0.2 0.2 0.2 0.2 phosphate hydrate Sodium chloride 0.39 0.39 0.390.39 0.39 0.39 Potassium chloride 0.15 0.15 0.15 0.15 0.15 0.15 Sodiumedetate 0.01 0.01 0.01 0.01 0.007 0.01 hydrate Polysorbate 80 0.00050.0005 0.0005 — — — Chlorhexidine 0.002 0.0025 0.002 0.0025 0.002 0.002gluconate pH 7.2 7.5 7.8 7.5 7.2 7.2

(Test Method)

The preservative effectiveness test was carried out in compliance withthe preservative effectiveness test method of the 16th edition of theJapanese Pharmacopoeia. For this test, the following test microorganismswere used: Escherichia coli (E. coli), Pseudomonas aeruginosa (P.aeruginosa), Staphylococcus aureus (S. aureus), Candida albicans (C.albicans) and Aspergillus brasiliensis (A. brasiliensis).

(Test Results)

The test results are shown in Table 2.

TABLE 2 Ophthalmic Ophthalmic Ophthalmic Ophthalmic OphthalmicOphthalmic Solution 1 Solution 2 Solution 3 Solution 4 Solution 5Solution 6 E. coli 2 weeks >4.3 >5.6 >4.3 >5.7 >4.6 >4.3 4weeks >4.3 >5.6 >4.3 >5.7 >4.6 >4.3 P. aeruginosa 2weeks >4.7 >5.8 >4.7 >5.6 >4.6 >4.7 4weeks >4.7 >5.8 >4.7 >5.6 >4.6 >4.7 S. aureus 2weeks >4.9 >6.0 >4.9 >5.6 >4.5 >4.9 4weeks >4.9 >6.0 >4.9 >5.6 >4.5 >4.9 C. albicans 2weeks >4.5 >5.7 >4.5 >5.4 >4.5 4.5 4 weeks >4.5 >5.7 >4.5 >5.4 >4.5 >4.5A. brasiliensis 2 weeks 1.7 1.3 1.7 1.0 0.7 1.4 4 weeks 2.4 1.7 2.5 1.70.9 1.5 Evaluation (JP) Acceptable Acceptable Acceptable AcceptableAcceptable Acceptable

Furthermore, the test results of Table 2 indicate the degree to whichthe number of viable organisms at the time of testing decreased incomparison with the number of inoculated organisms in terms of the logreduction, and a value of “1”, for example, indicates that the number ofviable organisms at the time of testing decreased by 10%.

As shown in Table 2, Ophthalmic Solutions 1 to 6 were shown to beacceptable based on the preservative effectiveness test standards of theJapanese Pharmacopoeia.

(Discussion)

On the basis of the above results, the present ophthalmic solution wasshown to have superior preservative effectiveness.

TEST EXAMPLE 2 Deformation Prevention Evaluation Test of Soft ContactLenses

The effects on soft contact lenses were examined using OphthalmicSolution 4.

(Sample Preparation)

Ophthalmic Solution 4 was prepared in the same manner as OphthalmicSolution 1 in accordance with the formulation shown in Table 1.

(Test Method)

Contact lenses corresponding to Group IV of the FDA classification ofcontact lenses shown in Table 3 (2-week Acuvue® Contact Lenses) wereimmersed in Ophthalmic Solution 4 for 24 hours followed by calculatingchanges in the diameter and base curve before and after immersion toexamine whether or not the evaluation criteria shown in the followingTable 4 are satisfied. In addition, the properties of each of thecontact lenses were observed following completion of testing.Furthermore, the evaluation criteria were established based on theContact Lens Standards for Vision Correction of Ministerial NotificationNo. 349 of the Ministry of Health, Labour and Welfare (Oct. 5, 2001).

TABLE 3 Classification¹⁾ Properties Group I Moisture content of lessthan 50%, nonionic Group II Moisture content of 50% or more, nonionicGroup III Moisture content of less than 50%, ionic Group IV Moisturecontent of 50% or more, ionic ¹⁾Classification of the U.S. Food and DrugAdministration (FDA)

(Evaluation Criteria)

TABLE 4 Diameter Tolerance of within ±0.20 mm of indicated diameter Basecurve Tolerance of within ±0.20 mm of indicated base curve PropertiesAbsence of internal air bubbles, impurities, discoloration, surfacedefects and surface irregularities, presence of smoothly rounded edges

(Results)

The results are shown in Table 5.

TABLE 5 Parameter Result Diameter (mm) +0.02 Base curve (mm) +0.02Properties No change Evaluation Satisfied standards

As shown in Table 5, the contact lens satisfied the evaluation criteriaafter being immersed for a long period of time. Accordingly, OphthalmicSolution 4 was shown to prevent deformation of soft contact lenses.

(Discussion)

Based on the above results, since the present ophthalmic solutionprevents deformation of soft contact lenses, it can be used for softcontact lenses.

TEST EXAMPLE 3 Cytotoxicity Test Using Corneal Epithelial Cells

A cytotoxicity test using corneal epithelial cells was carried out toexamine the effects of the present ophthalmic solution on cornealepithelial cells.

(Sample Preparation)

Ophthalmic Solutions 7, 8, 9 and 10 were prepared in the same manner asOphthalmic Solution 1 in accordance with the formulations shown in Table6.

TABLE 6 (Units: g/100 ml) Oph- Oph- Oph- Oph- thalmic thalmic thalmicthalmic Solu- Solu- Solu- Solu- tion 7 tion 8 tion 9 tion 10 Diquafosolsodium 3 — 3 — Sodium hydrogen phosphate 0.2 0.2 0.2 0.2 hydrate Sodiumchloride 0.39 0.39 0.41 0.41 Potassium chloride 0.15 0.15 0.15 0.15Sodium edetate hydrate 0.01 0.01 0.01 0.01 Benzalkonium chloride¹⁾ — —0.002 0.002 Chlorhexidine gluconate 0.0025 0.0025 — — pH 7.5 7.5 7.5 7.5¹⁾BAK, chemical structural formula: [C₆H₅CH₂N(CH₃)₂C₁₂H₂₅]Cl

(Test Method)

SV40 immortalized human corneal epithelial cells (HCE-T: Riken,Bioresource Center, Cell No. RCB2280) were inoculated into a 96-wellplate (1×10⁴ cells/well) and cultured for 1 day in D-MEM/F12 mediumcontaining 10% FBS. On the following day, the medium was replaced withOphthalmic Solution 7, Ophthalmic Solution 8, Ophthalmic Solution 9 orOphthalmic Solution 10 followed by culturing the aforementioned cornealepithelial cells for 15 minutes. Cell viability (equivalent toabsorbance of 490 nm) was measured using a Cell Proliferation Assay Kit(Promega Corp., Catalog No.: G3580).

(Results)

The test results are shown in FIG. 1.

As is clear from FIG. 1, the diquafosol ophthalmic solution comprisingchlorhexidine gluconate (Ophthalmic Solution 7) demonstrated a higherdegree of cell viability in cultured immortalized human cornealepithelial cells than ophthalmic solutions comprising BAK (OphthalmicSolutions 9 and 10) and the ophthalmic solution comprising chlorhexidinebut not comprising diquafosol or a salt thereof (Ophthalmic Solution 8).

(Discussion)

Since the present ophthalmic solution demonstrates a high degree of cellviability in cultured immortalized human corneal epithelial cells, it ishighly safe with respect to a living body, and particularlykeratoconjunctival epithelium, and is useful for diseases such as dryeye in which the keratoconjunctival epithelium is unstable.

TEST EXAMPLE 4 Evaluation Test of NIBUT Increasing Effect 1

The NIBUT values of a diquafosol ophthalmic solution were examined ineyes in which tear film stability had decreased as a result of wearingsoft contact lenses.

(Sample Preparation)

Ophthalmic Solution 4 was prepared in the same manner as OphthalmicSolution 1 in accordance with the formulation shown in Table 1.

(Test Method)

NIBUT values before and 15, 30, 45 and 60 minutes after applyingOphthalmic Solution 4 (20 μl/eye) were measured for the eyes ofcynomolgus monkeys wearing soft contact lenses (product name: MeniconSoft MA®) with a dry eye observation system (DR-1, Kowa Co., Ltd.). Anartificial tear solution (product name: Soft Santia®) was used as acontrol (N=10 to 11 eyes).

(Results)

The test results are shown in FIG. 2. As is clear from FIG. 2, whenOphthalmic Solution 4 was applied to the eyes wearing soft contactlenses, significant increases in NIBUT were observed in comparison withprior to application at all measurement points up to 60 minutes afterapplication. On the other hand, increases in NIBUT were not observed ineyes applied with the artificial tear solution.

(Discussion)

On the basis of the above results, the present ophthalmic solution wasshown to improve decreases in tear film stability caused by wearing softcontact lenses. This effect of the present ophthalmic solution wasprominent even when compared with an artificial tear solution typicallyused for the treatment of dry eye. Accordingly, the present ophthalmicsolution is useful for the prevention and/or treatment of dry eye in aneye wearing a soft contact lens. In addition, the present ophthalmicsolution is also useful for the prevention and/or treatment of eyedryness and/or eye discomfort in an eye wearing a soft contact lens.

TEST EXAMPLE 5 Evaluation Test of NIBUT Increasing Effect 2

The NIBUT values of a diquafosol ophthalmic solution were examined ineyes in which tear film stability had decreased as a result of wearingsoft contact lenses.

(Sample Preparation)

Ophthalmic Solution 4 was prepared in the same manner as OphthalmicSolution 1 in accordance with the formulation shown in Table 1.

(Test Method)

NIBUT values before and 5, 15, 30, 45 and 60 minutes after applyingOphthalmic Solution 4 (20 μl/eye) were measured for the eyes ofcynomolgus monkeys wearing soft contact lenses (product name: MeniconSoft MA®) with a dry eye observation system (DR-1, Kowa Co., Ltd.). Anartificial tear solution (product name: Soft Santia®) and sodiumhyaluronate (product name: Hyalein® Mini Ophthalmic Solution 0.1%) wasused as controls (N=11 eyes).

(Results)

The test results are shown in FIG. 3. As is clear from FIG. 3, whenOphthalmic Solution 4 was applied to the eyes wearing soft contactlenses, significant increases in NIBUT were observed in comparison withprior to application at all measurement points up to 60 minutes afterapplication. On the other hand, increases in NIBUT were not observed ineyes applied with the artificial tear solution. In addition, although anincrease in NIBUT was observed 5 minutes after application in eyesadministered sodium hyaluronate, the increasing effect thereof was lowerthan that of Ophthalmic Solution 4 and increases in NIBUT were notobserved at 15 minutes after application and beyond.

(Discussion)

On the basis of the above results, the present ophthalmic solution wasshown to improve decreases in tear film stability caused by wearing softcontact lenses. This effect of the present ophthalmic solution wasprominent even when compared with an artificial tear solution and asodium hyaluronate ophthalmic solution typically used for the treatmentof dry eye. Accordingly, the present ophthalmic solution is useful forthe prevention and/or treatment of dry eye in an eye wearing a softcontact lens. In addition, the present ophthalmic solution is alsouseful for the prevention and/or treatment of eye dryness and/or eyediscomfort in an eye wearing a soft contact lens.

TEST EXAMPLE 6 Comparative Test of NIBUT Increasing Effect

A comparative study of NIBUT values was conducted between the presentophthalmic solution and an ophthalmic solution comprising BAK(ophthalmic solution comprising diquafosol sodium and BAK) in eyes inwhich tear film stability had decreased as a result of wearing softcontact lenses.

(Sample Preparation)

Ophthalmic Solution 4 was prepared in the same manner as OphthalmicSolution 1 in accordance with the formulation shown in Table 1.

In addition, Ophthalmic Solution 11 comprising BAK instead of thechlorhexidine gluconate of Ophthalmic Solution 4 was prepared for use asa comparative example. More specifically, diquafosol sodium (3 g),sodium hydrogen phosphate hydrate (0.2 g), sodium chloride (0.41 g),potassium chloride (0.15 g) and BAK (0.0075 g) were dissolved in waterand brought to a final volume of 100 mL followed by the addition of a pHadjuster to the adjust the pH to 7.5. Ophthalmic Solution 4 andOphthalmic Solution 11 are ophthalmic solutions that both comprise thesame concentrations of active ingredient (diquafosol sodium). Inaddition, Ophthalmic Solution 4 and Ophthalmic Solution 11 areophthalmic solutions that both comply with the preservativeeffectiveness test standards of the Japanese Pharmacopoeia and haveequivalent preservative effectiveness.

(Test Method)

NIBUT values before and 30 minutes after applying Ophthalmic Solution 4or Ophthalmic Solution 11 (20 μl/eye) were measured for the eyes ofcynomolgus monkeys wearing soft contact lenses (product name: MeniconSoft MA®) with a dry eye observation system (DR-1, Kowa Co., Ltd.) (N=11eyes).

(Results)

The test results are shown in Table 7.

TABLE 7 Ophthalmic Ophthalmic Solution 4 Solution 11 NIBUT beforeapplication (sec) 3.40 3.39 NIBUT 30 minutes after application (sec)8.20 6.25

As a result of measuring and comparing NIBUT values before and 30minutes after applying the present ophthalmic solution (OphthalmicSolution 4) or a BAK-comprising ophthalmic solution (Ophthalmic Solution11), which comply with the preservative effectiveness test standards ofthe Japanese Pharmacopoeia and have equivalent preservativeeffectiveness, the present ophthalmic solution was shown to have higherNIBUT increasing effect than the BAK-comprising ophthalmic solution.

(Discussion)

Based on the above results, the present ophthalmic solution was shown toimprove decreases in tear film stability caused by wearing soft contactlenses to a greater degree than an ophthalmic solution comprising BAK.

PREPARATION EXAMPLES

Although the following provides a more detailed explanation ofpreparations of the present invention by indicating examples thereof,the present invention is not limited to these preparation examples.

Formulation Example 1 Ophthalmic Solution (3% (w/v))

In 100 ml Diquafosol sodium 3 g Sodium hydrogen phosphate hydrate0.1-0.5 g Sodium chloride 0.01-1 g Potassium chloride 0.01-1 g Sodiumedetate hydrate 0.0001-0.1 g Chlorhexidine gluconate 0.0001-0.1 gSterile purified water q.s.Diquafosol sodium and other ingredients listed above are added tosterile purified water and they are mixed sufficiently so that thisophthalmic solution can be prepared.

Formulation Example 2 Ophthalmic Solution (3% (w/v))

In 100 ml Diguasofol sodium 3 g Sodium hydrogen phosphate hydrate0.1-0.5 g Sodium chloride 0.01-1 g Potassium chloride 0.01-1 g Sodiumedetate hydrate 0.0001-0.1 g Chlorhexidine gluconate 0.0001-0.1 gPolysorbate 80 0.0001-0.1 g Sterile purified water q.s.Diquafosol sodium and other ingredients listed above are added tosterile purified water and they are mixed sufficiently so that thisophthalmic solution can be prepared.

INDUSTRIAL APPLICABILITY

The present ophthalmic solution has superior preservative effectiveness.Moreover, since the present ophthalmic solution prevents deformation ofsoft contact lenses, it can also be used for soft content lenses. Inaddition, the present ophthalmic solution demonstrates higher cellviability in cultured immortalized human corneal epithelial cells thanophthalmic solutions comprising BAK and ophthalmic solutions comprisingchlorhexidine but not comprising diquafosol or a salt thereof.Accordingly, the present ophthalmic solution is highly safe with respectto a living body, and particularly keratoconjunctival epithelium, and isuseful for diseases such as dry eye in which keratoconjunctivalepithelium is unstable. In addition, the present ophthalmic solutionsignificantly increases NIBUT in an eye wearing a soft contact lens. Onthe other hand, this effect is not observed with artificial tearsolutions. Namely, the present ophthalmic solution stabilizes the tearfilm in an eye wearing a soft contact lens. Since the onset and/orexacerbation of dry eye symptoms caused by wearing soft contact lensesis attributable to a decrease in tear film stability, stabilization ofthe tear film by the present ophthalmic solution is useful for theprevention and/or treatment of dry eye in an eye wearing a soft contactlens. In addition, the present ophthalmic solution is also useful forthe prevention and/or treatment of eye dryness and/or eye discomfort inan eye wearing a soft contact lens.

The invention claimed is:
 1. A method for prevention and/or treatment ofdry eye, comprising application of an aqueous ophthalmic solutioncomprising diquafosol or a salt thereof at a concentration of 0.1% to10% (w/v) and a chlorhexidine at a concentration of 0.002% to 0.005%(w/v) to an eye of a patient.
 2. The method according to claim 1,wherein the chlorhexidine is chlorhexidine gluconate.
 3. The methodaccording to claim 1, wherein a concentration of diquafosol or a saltthereof in the ophthalmic solution is 3% (w/v).
 4. The method accordingto claim 1, wherein the aqueous ophthalmic solution further comprises achelating agent.
 5. The method according to claim 4, wherein thechelating agent is edetic acid or a salt thereof.
 6. The methodaccording to claim 1, wherein the aqueous ophthalmic solution is for asoft contact lens.
 7. The method according to claim 6, wherein the softcontact lens is a silicone hydrogel contact lens.
 8. The methodaccording to claim 1, wherein the dry eye is caused by wearing a softcontact lens.